1. Field of the Invention
The present invention is directed to recombinant microalgal cells and their use in production of hemagglutinin-neuraminidase (HN) polypeptides, as well as compositions and uses thereof.
2. Background
Production of proteins via the fermentation of microorganisms presents several advantages over existing systems such as plant and animal cell culture. For example, microbial fermentation-based processes can offer (i) rapid production of high concentration of protein; (ii) the ability to use sterile, well-controlled production conditions (such as Good Manufacturing Practice (GMP) conditions); (iii) the ability to use simple, chemically defined growth media allowing for simpler fermentations and fewer impurities; (iv) the absence of contaminating human or animal pathogens; and (v) the ease of recovering the protein (e.g., via isolation from the fermentation media). In addition, fermentation facilities are typically less costly to construct than cell culture facilities.
Microalgae, such as thraustochytrids, can be grown with standard fermentation equipment, with very short culture cycles (e.g., 1-5 days), inexpensive defined media and minimal purification, if any. Furthermore, certain microalgae, e.g., Schizochytrium, have a demonstrated history of safety for food applications of both the biomass and lipids derived therefrom. For example, DHA-enriched triglyceride oil from this microorganism has received GRAS (Generally Recognized as Safe) status from the U.S. Food and Drug Administration.
Microalgae have been shown to be capable of expressing recombinant proteins. For example, U.S. Pat. No. 7,001,772 discloses the first recombinant constructs suitable for transforming thraustochytrids, including members of the genus Schizochytrium. This publication discloses, among other things, Schizochytrium nucleic acid and amino acid sequences for an acetolactate synthase, an acetolactate synthase promoter and terminator region, an α-tubulin promoter, a promoter from a polyketide synthase (PKS) system, and a fatty acid desaturase promoter. U.S. Publ. Nos. 2006/0275904 and 2006/0286650, subsequently discloses Schizochytrium sequences for actin, elongation factor 1 alpha (ef1α), and glyceraldehyde 3-phosphate dehydrogenase (gapdh) promoters and terminators.
Viral vaccines are often made from inactivated or attenuated preparations of viral cultures corresponding to the disease they are intended to prevent. Generally, a virus is cultured from the same or similar cell type as the virus might infect in the wild. Such cell culture is expensive and often difficult to scale. To address this problem, attempts have been made to express viral protein antigens in transgenic hosts, which can be less costly to culture and more amenable to scale. However, viral membrane proteins such as hemagglutinin-neuraminidase (HN) protein can be very difficult to produce in large amounts, and attempts to express viral envelope proteins in whole or in part in heterologous systems have often been met with limited success. Thus, there is a need for new heterologous expression systems, such as those of the present invention, that are scaleable and able to produce viral HN antigens.